| Pine pollen polysaccharides and sulfated polysaccharides have good immune activity and play an important role in regulating inflammatory factors.The previous research in our laboratory confirmed that pine pollen polysaccharides and sulfated polysaccharides have therapeutic effects on ulcerative colon in mice at immune level,and speculated whether the regulation of pine pollen polysaccharides and sulfated polysaccharides on immune factors is related to protecting intestinal epithelial cell barrier,and a great deal of studies have found that necroptosis is closely related to inflammation.In this experiment,the methods of extraction,purification and esterification of polysaccharides in the previous laboratory were used.On the basis of exploring the modeling method of UC mice,the effects of pine pollen polysaccharides and sulfated polysaccharides on regulating intestinal barrier and whether they can play an anti-inflammatory role in vivo by regulating RIPK3 dependent necroptosis pathways were mainly explored.This experiment mainly includes the following parts:1.Extraction,purification and sulfation of polysaccharides from pine pollen.The extraction of polysaccharides from pine pollen followed the previous extraction method of this laboratory.The method of water boiling and alcohol precipitation was used for extraction,and trichloroacetic acid was used for deproteinization.According to the yield and purity,the polysaccharide extracted from 60%ethanol was finally selected,which was called PPM60.PPM60 was separated and purified by Sephacryl TMS-400,and was divided into five components.According to the yield,the third component was selected to continue the follow-up experiment,named PPM60-Ⅲ.Sulfated PPM60-Ⅲby chlorosulfonic acid-pyridine method to obtain sulfated polysaccharide SPPM60-Ⅲ.2.According to the previous research results in the laboratory,the animal model of ulcerative colitis was established with 3%DSS for 7 days.They were divided into four groups,named,normal group（HC）,model group（DSS）,polysaccharide group（PPM）and sulfated polysaccharide group（SPPM）.Weigh the body weight every day,observe the fecal condition and record it.After the experiment,blood was collected under anesthesia,and the weight change map,DAI score map and colon length map of mice were made.The pathological sections of colon were observed by HE staining.The weight loss of DSS mice was obvious,DAI score was obviously increased,colon length was obviously shortened,and colon injury was serious.The change trend was alleviated in PPM and SPPM groups,and colon injury was repaired in different degrees.3.Elisa test detected inflammatory factors in mouse serum,including IL-10,IL-18,IL-6,TNF-αand IL-1β.The changes of COX-2 and i NOS in colon tissue were detected by Western blotting experiment.The results showed that in DSS group,the content of anti-inflammatory factor IL-10decreased,and the content of pro-inflammatory factors IL-18,IL-6,TNF-αand IL-1βincreased,while PPM and SPPM groups significantly improved this situation.In DSS group,the content of induced enzyme increased,while PPM group and SPPM group significantly decreased the content of induced enzyme.4.To study the expression changes of RIPK3 pathway related proteins in mice.In this thesis,Western blotting was used to analyze the content changes of the marker proteins of RIPK3pathway,including RIPK3,MLKL,RIPK1 and Caspase-8,and further analyze the expression of the marker proteins of phosphorylation in this pathway,including p-RIPK3,p-RIPK1 and p-MLKL.The experimental results showed that the protein expressions of RIPK3,MLKL and RIPK1 increased in DSS-induced colitis mice,but decreased in PPM and SPPM groups,which indicated that RIPK3,MLKL and RIPK1 played a role in treating inflammation and regulated RIPK3 pathway.In addition,the expressions of p-RIPK3,p-RIPK1 and p-MLKL in DSS-induced colitis mice increased significantly,while the expression of Caspase-8 decreased in PPM and SPPM groups,which indicated that the programmed necroptosis pathway mediated by RIPK3 might be regulated in this process to treat mouse colitis.In addition,the results of RT-q PCR showed the same trend,but there were no significant difference.5.To study the changes of tight junction of intestinal epithelial cells in mouse colon tissue.The tight junction of intestinal epithelial cells was observed by transmission electron microscope,the expressions of tight junction related proteins ZO-1 and Occludin were analyzed by immunofluorescence,and the expressions of ZO-1,Occludin and Claudin-1 were analyzed by Western blotting.The results showed that the tight junction of intestinal epithelial cells in mice with colitis induced by DSS was destroyed and the expression of tight junction related proteins decreased,while the tight junction of cells in PPM and SPPM groups was improved and the expression of related proteins increased.6.In this experiment,16S r DNA amplicon sequencing technology was used to study the diversity of microbial population in mouse colon contents.The results showed that Firmicutes,Bacteroidetes and Proteobacteria were the dominant bacteria of intestinal flora,among which the abundance of Firmicutes and Bacteroidetes decreased in DSS model group,while Proteobacteria increased in DSS model group.PPM and SPPM groups improved their changes.The abundance of harmful bacteria such as Gammaproteobacteria,Bacilli,Escherichia-Shigella were higher in DSS group,while the abundance of beneficial bacteria such as Bacteroides,Alloprevotella,Akkermansia and Faecalibacterium decreased.The PPM and SPPM groups adjusted the abundance of bacteria,improved the change of flora and maintained the balance of intestinal flora.The regulation of flora has further restored the barrier protection of epithelial cells.The experimental results showed that PPM60-III and SPPM60-III had certain therapeutic effects on UC mice,and the mechanism may be through regulating RIPK3 pathway and adjusting the balance of intestinal flora to further restore the tight junction of epithelial cells to regulate inflammation. |
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